5-氮胞苷诱导骨髓间充质干细胞向心肌细胞的分化  

Marrow mesenchymal stem cells differentiating into myocardial cells induced by 5-azacytidine

作　　者：郭茂娟[1] 范英昌[2] 徐秀梅[1] 赵旭[1] 

机构地区：[1]天津中医药大学,2005级天津市300193 [2]天津中医药大学病理教研室,天津市300193

出　　处：《中国组织工程研究与临床康复》2007年第46期9238-9241,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：天津市社会发展科技计划项目(043111211)~~

摘　　要：目的:目前在5-氮胞苷诱导骨髓间充质干细胞向心肌细胞分化领域中,对用于诱导的细胞代次选择不一。实验选择第2,9代骨髓间充质干细胞,观察比较经5-氮胞苷体外诱导后其各自向心肌细胞分化过程中心肌特异性肌钙蛋白cTnT和早期分化基因的表达。方法:实验于2005-07/2007-07在天津中医药大学病理三级实验室完成。①实验方法:清洁级Wistar雄性大鼠,脱颈处死后取双侧股骨、胫骨,采用密度梯度离心法分离骨髓单个核细胞进行原代培养。去掉原代细胞瓶内的培养液,D-Hank’s液冲洗去除血清,胰蛋白酶+乙二胺四乙酸消化,制备单细胞悬液,按1∶3传代。用10μmol/L的5-氮胞苷分别诱导第2,9代骨髓间充质干细胞,诱导4周,并设立未加诱导剂的阴性对照。②实验评估:倒置相差显微镜下观察细胞的生长情况及形态变化;免疫组织细胞化学法检测心肌特异性肌钙蛋白cTnT的表达,胞浆出现棕黄色颗粒状物质为阳性;嵌合荧光法检测心肌早期基因NKx2.5、GATA-4、Desmin、α-actin的相对表达量。结果:①细胞形态学观察:5-氮胞苷诱导前细胞生长较快,诱导后死亡细胞较多且生长较慢。诱导后2,3,4周,第9代骨髓间充质干细胞无论在形态和细胞活力方面均优于第2代。②心肌特异性肌钙蛋白cTnT的表达:5-氮胞苷诱导4周后,第9代骨髓间充质干细胞心肌特异性肌钙蛋白cTnT阳性表达率明显高于第2代(22.42±9.97),(11.22±5.62)%,P<0.05,阴性对照无阳性表达。③心肌早期基因的表达:反转录-聚合酶链反应结果显示,5-氮胞苷诱导4周后,第9代骨髓间充质干细胞心肌早期基因NKx2.5、GATA-4、Desmin、α-actin的相对表达量均明显高于第2代(P<0.05)。结论:经5-氮胞苷诱导后,骨髓间充质干细胞表达心肌特异性肌钙蛋白cTnT及4种心肌早期基因,证实其能够向心肌细胞分化,且传至第9代时向心肌细胞分化的能力要强于第2代。AIM： In the domain of inducing marrow mesenchymal stem cells （MSCs） into myocardial cells with 5-azacytidine, there is a problem about the choose of induced cells＇ generation. The second （P2） and the ninth （P9） generation were chosen to experimentize, and to observe the expression of Cardiac Specific protein Troponin T （cTnT） and early differentiation genes induced by 5-azacytidine. METHODS： The experiment was performed in Pathology Laboratory of Tianjin University of Traditional Chinese Medicine from July 2005 to July 2007.①Experimental methods： Depuratory class Wistar male rats were sacrificed and took out of femurs and Tibials. Density gradient centrifugation separation was used to separate primary MSCs. Culture solution were removed and then the cells were clean with D-Hank＇s, then digested with trypsin and ethylenediamine tetraacetic acid （EDTA） to prepare single cell suspensions to passage as 1：3 proportion. P2 and P9 MSCs were induced into myocardial cells with 10 μmol/L 5-azacytidine for 4 weeks, and negative control group was set up. ②Experimental evaluation： During the procedure of inducing the P2 and P9 MSCS, the change of the cell morphology was observed. Expression of cTnT was detected with immunocytochemical method. Positive cells were expressed in cytoplasm. Myocardial early gene was measured with reverse transcription-polymerase chain raction （RT-PCR） such as NKx2.5, GATA-4, Desmin and α-actin. RESULTS： ①Cell morphological observation： Cells grew rapidly before 5-azacytidine induction, but more cells died and grew slowly after inducing. P9 was prior to P2 not only on cell morphology change but also on ceil viability at weeks 2, 3 and 4 after inducing. ②Expression of cTnT： Inducing MSCs into myocardial cells with 5-azacytidine after four weeks, the relative amount of cTnT P9 was more than P2 [（22.42±9.97）, （11.22±5.62）% P 〈 0.05], and no expression was found in the control group. ③Early differentiation genes： Result of RT-PCR s

关 键 词：5-氮胞苷 诱导分化 骨髓间充质干细胞 

分 类 号：R394.2[医药卫生—医学遗传学]